CN107058110B - A kind of preservation and recovery method of Haematococcus pluvialis cells - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract 16
- 241000168517 Haematococcus lacustris Species 0.000 title claims abstract 13
- 238000004321 preservation Methods 0.000 title claims abstract 8
- 238000011084 recovery Methods 0.000 title claims abstract 8
- 238000007710 freezing Methods 0.000 claims abstract 3
- 230000008014 freezing Effects 0.000 claims abstract 3
- 230000022472 cold acclimation Effects 0.000 claims abstract 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims 4
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims 3
- 229910052757 nitrogen Inorganic materials 0.000 claims 2
- 239000012141 concentrate Substances 0.000 claims 1
- 238000005286 illumination Methods 0.000 claims 1
- 241000195493 Cryptophyta Species 0.000 abstract 5
- 241000894006 Bacteria Species 0.000 abstract 3
- 230000004083 survival effect Effects 0.000 abstract 3
- 230000007774 longterm Effects 0.000 abstract 2
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 241000894007 species Species 0.000 abstract 1
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Abstract
本发明属于藻类生物技术领域，具体涉及一种雨生红球藻细胞的保存及复苏方法。将雨生红球藻细胞浓缩液经0～10摄氏度冷驯化后，在遮光条件、冰点以下冰冻保存。本发明通过将浓缩藻液经短时冷驯化后冰冻保存，而实现雨生红球藻细胞的高存活率的中长期保存方法；同时对保存细胞的复苏方法；本发明方法的复苏存活率高，在保存75天复苏后细胞存活率达到95％以上，复苏后生长迅速；有效避免细菌和杂藻繁殖，在长期保存阶段无明显的细菌和杂藻生长，复苏后细胞快速生长，抑制细菌和杂藻增殖；本发明方法简单，不需要借助专业设备；本发明方法易于大量藻种的保存，很容易在生产实践中实施。The invention belongs to the technical field of algae, and in particular relates to a preservation and recovery method of Haematococcus pluvialis cells. After the concentrated solution of Haematococcus pluvialis cells is acclimated by cold at 0-10 degrees Celsius, it is frozen and stored under shading conditions and below freezing point. The present invention realizes a medium- and long-term preservation method with high survival rate of Haematococcus pluvialis cells by freezing and storing the concentrated algal liquid after short-term cold acclimation; meanwhile, a method for revitalizing the preserved cells; the method of the invention has a high recovery survival rate , after 75 days of storage, the cell survival rate reaches more than 95%, and the growth is rapid after recovery; effectively avoiding the reproduction of bacteria and miscellaneous algae, there is no obvious growth of bacteria and miscellaneous algae in the long-term storage period, and the cells grow rapidly after recovery, inhibiting bacteria and miscellaneous algae. Proliferation of miscellaneous algae; the method of the present invention is simple and does not require professional equipment; the method of the present invention is easy to preserve a large number of algal species, and is easy to implement in production practice.
Description
Technical Field
The invention belongs to the technical field of algae biology, and particularly relates to a preservation and recovery method of haematococcus pluvialis cells.
Background
Haematococcus pluvialis (Haematococcus pluvialis) is a green alga that produces natural astaxanthin and belongs to the family Haematococcus of the order Volvocales of the class Chlorophyceae of the phylum Chlorophyta. The cells generate a large amount of astaxanthin under specific environment and store the astaxanthin in the cells, and the content of the astaxanthin is up to 7 percent. Astaxanthin in haematococcus pluvialis is mainly esterified astaxanthin, and the molecular structure is 3S, 3S' configuration, so that astaxanthin is considered as the best natural astaxanthin source, has wide application in the fields of health food, coloring agents, personal care products and high-end feeds, and becomes a hot spot for resource development and utilization of algae in recent years.
In the large-scale production process of haematococcus pluvialis, a large number of high-activity cells are needed as starting algae species in an outdoor production stage. Currently, a large amount of starting algal species is obtained mainly by multi-stage cultivation, and thus it takes much time and takes much culture volume to produce the starting algal species before mass production. In actual production practice, on the one hand, due to the high culture failure rate of the starting provenance, especially the green zoospore, due to mass death caused by algae-eating organism pollution or incapability of tolerating high environmental light, the supply of the generated starting provenance is insufficient so as to limit the production of the next stage; on the other hand, continuous production cannot be carried out in parts of China due to the limitation of seasons and climates, so that production in the next year is easily delayed by the consumption of the culture time of the starting provenance, and particularly, the production is serious when the climatic conditions are unfavorable for the culture of the starting algae. The method for preserving the high-activity cells is an effective method for guaranteeing successful and stable production by timely and sufficiently providing the high-density and high-activity initial algae species which can be rapidly rejuvenated and can be rapidly proliferated, is directly related to the benefit of astaxanthin production, and becomes one of bottleneck problems for restricting the development of haematococcus pluvialis astaxanthin.
At present, microalgae preservation methods comprise methods such as subculture preservation, dry preservation, concentrated solution low-temperature preservation, ultralow-temperature freezing preservation and the like. The subculture method is the most widely used traditional method, including liquid and immobilized subculture methods, and has the advantage of simple operation, but has the problems of rapid nutrient salt consumption, easy cell aging and the like, the culture medium needs to be replaced every other month, and the workload of frequent subculture is large. Through the conversion of light by the red membrane, the aging of haematococcus pluvialis vegetative cells can be slowed down to prolong the storage time (CN 103695314A-a method for storing haematococcus pluvialis vegetative cells), but the pollution of bacteria and other algae cannot be avoided in the practice of mass storage. The concentrated solution cryopreservation method is suitable for some unicellular algae, realizes high-activity preservation for about 3 months, but is easy to cause bacterial and miscellaneous algae pollution (CN 105838611A-a preservation method of chlorella concentrated solution; CN 105713838A-a high-activity cell preservation method of pseudonandina). The dry preservation method is only suitable for some filamentous algae (CN 105483006A-preservation method of filamentous blue algae; CN 103621395A-embedding dehydration ultralow-temperature preservation method of tawny algae filamentous body) and has low survival rate after recovery. Cryopreservation is a novel cell preservation method, has the advantages of keeping germplasm genetic stability and realizing long-term preservation for years. For example, haematococcus pluvialis can be preserved for a long time at a low temperature of liquid nitrogen by glycerol protection (CN105779292A — a cryopreservation method of haematococcus pluvialis subjected to aseptic treatment), but the survival rate of resuscitation is only 66.13%, and complicated operation techniques and expensive preservation equipment are required. Since the above method is limited in terms of cell survival rate, storage time, labor intensity, equipment requirements, etc., the efficiency and scale of storage are limited, thereby bringing many difficulties to practical production.
Disclosure of Invention
The invention aims to provide a method for preserving and recovering haematococcus pluvialis cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preservation method of haematococcus pluvialis cells comprises the steps of carrying out cold acclimation on a haematococcus pluvialis cell concentrated solution at 0-10 ℃, and then freezing and preserving under a light shielding condition and below a freezing point.
And culturing the haematococcus pluvialis cells to a chlamydospore state, collecting, and concentrating until the cell density is 10-200 g/L.
And (3) placing the collected haematococcus pluvialis chlamydospore state cell concentrated solution at 0-10 ℃ for freezing and acclimation for 1-5 days.
The freezing and acclimating temperature is 1-5 ℃.
And storing the domesticated algae liquid for 3-5 months at-20 to-1 ℃ under shading conditions.
A recovery method of haematococcus pluvialis cells stored by a storage method is characterized in that haematococcus pluvialis chlamydospore cells subjected to cold acclimation treatment and freezing and stored at low temperature and in a dark place are unfrozen at the temperature of 5-35 ℃, then a nitrogenous culture medium is added for dilution, and then the haematococcus pluvialis chlamydospore cells are cultured under the illumination condition to obtain the recovery of the cells.
The content of nitrogen element in the nitrogen-containing culture medium is 0-200 mg/L.
The content of nitrogen elements in the nitrogen-containing culture medium is 50-100 mg/L.
The diluted cell concentration is 0.2-1.0 g/L.
Compared with the prior art, the invention has the following advantages:
the invention realizes the medium-long term preservation method of high survival rate of haematococcus pluvialis cells by freezing and preserving the concentrated algae solution after short-time cold acclimation; a method for simultaneously recovering the preserved cells; the method specifically comprises the following steps:
1. the recovery survival rate of the method is high, the cell survival rate reaches more than 95 percent after 75 days of recovery, and the cell grows rapidly after recovery;
2. the propagation of bacteria and hybrid algae is effectively avoided, no obvious bacteria and hybrid algae grow in the long-term storage stage, cells grow rapidly after recovery, and the proliferation of the bacteria and the hybrid algae is inhibited;
3. the method is simple and does not need to use professional equipment;
4. the method of the invention is easy for preserving a large amount of algae species and is easy to implement in production practice.
Detailed Description
The technical scheme of the invention is described in detail by combining part of the embodiments. The described embodiments are only a part, not all of the embodiments of the invention.
Example 1
Haematococcus pluvialis NIES-144 strain (commercially available) was prepared in the following manner: 1) in total nitrogen BBM medium (commercially available) at 50. mu.E/m2Culturing the strain under illumination for 5 days to obtain green zoospore;
2) 300 μ E/m in nitrogen-free BBM medium2Culturing the strain under illumination for 10 days to obtain chlamydospores;
standing, settling and concentrating the obtained green zoospores and chlamydospores, and then respectively putting the zoospores and the chlamydospores into a refrigerator fresh-keeping chamber for cold acclimation at 5 ℃ for 24 hours; then transferring to a refrigerating chamber to freeze at-20 ℃ to ice and continuously freezing for 60 days; taking out after freezing and thawing at room temperature.
Then, the melted concentrated algae solution is respectively diluted by BBM culture medium containing 4mmol/L sodium nitrate until the cell density is 0.449g/L, and the diluted concentrated algae solution is placed at 200 muE/m2S lightIrradiating with 1% CO2Air culture of (2). After 48 hours of illumination culture, observing and counting the proportion of the cells capable of exciting the red chlorophyll fluorescence by using a fluorescence microscope, and counting to obtain that the survival rates of the green zoospores and the chlamydospores are respectively 51.3 percent and 97.5 percent; then, the culture is continued for 8 days under the conditions, the cell density of the green zoospore and the chlamydospore respectively reaches 0.668 g/L and 2.768g/L, and the specific growth rates of the green zoospore and the chlamydospore are respectively calculated to be 0.05 and 0.227.
Comparative example 1
Haematococcus pluvialis NIES-144 strains, respectively: 1) 50 μ E/m in total nitrogen BBM medium2Culturing the strain under illumination for 5 days to obtain green zoospore;
2) 300 μ E/m in nitrogen-free BBM medium2Culturing the strain under illumination for 10 days to obtain chlamydospores;
respectively freezing the obtained green zoospores and chlamydospores into ice at-20 ℃ in a refrigerating chamber of a refrigerator and continuously freezing for 60 days; then taking out and melting at room temperature.
Then, the melted concentrated algae solution is respectively diluted by BBM culture medium containing 4mmol/L sodium nitrate until the cell density is 0.471g/L, and the diluted concentrated algae solution is placed at 200 mu E/m2Illumination is carried out per second and 1% CO is introduced2Air culture of (2). After 48 hours of illumination culture, observing and counting the proportion of the cells capable of exciting the red chlorophyll fluorescence by using a fluorescence microscope, and counting to obtain that the survival rates of the green zoospores and the chlamydospores are respectively 0.2 percent and 4.6 percent; then, the culture is continued for 8 days under the conditions, the cell density of the green zoospore and the sclerite reaches 0.512 and 0.891g/L respectively, and the specific growth rates of the green zoospore and the sclerite are calculated to be 0.01 and 0.079 respectively.
Comparative example 2
Haematococcus pluvialis NIES-144 strains, respectively: 1) 50 μ E/m in total nitrogen BBM medium2Culturing the strain under illumination for 5 days to obtain green zoospore;
2) 300 μ E/m in nitrogen-free BBM medium2Culturing the strain under illumination for 10 days to obtain chlamydospores;
green obtained as described aboveThe chromospore and the chlamydospore are respectively stored for 60 days at room temperature; then, the concentrated algae solution stored at room temperature is respectively diluted by BBM culture medium containing 4mmol/L sodium nitrate until the cell density is 0.510g/L, and the diluted concentrated algae solution is placed at 200 muE/m2Illumination is carried out per second and 1% CO is introduced2Air culture of (2). After 48 hours of illumination culture, observing and counting the proportion of the cells capable of exciting the red chlorophyll fluorescence by using a fluorescence microscope, and counting to obtain that the survival rates of the green zoospores and the chlamydospores are 57.3 percent and 75.2 percent respectively; then, the culture is continued for 8 days under the conditions, the cell density of the green zoospore and the chlamydospore respectively reaches 1.730 and 1.323g/L, and the specific growth rates of the green zoospore and the chlamydospore are calculated to be 0.15 and 0.11 respectively.
Comparing the cell viability and specific growth rate after cell preservation recovery in example 1 with the two comparative examples, the effect of cell morphology differences on viability and specific growth rate and bacterial concentration in cell culture is shown in table 1:
TABLE 1
The data show that the survival rate of the haematococcus pluvialis cells which are subjected to cold acclimation and then are subjected to freezing preservation is obviously higher than that of the haematococcus pluvialis cells which are subjected to direct freezing preservation, and the growth speed and the specific growth rate are obviously higher than those of the haematococcus pluvialis cells subjected to direct freezing preservation. Microscopic observation shows that the probability of the cultured contamination after the recovery of the frozen and preserved cells is very low no matter whether the cells are subjected to cold acclimation treatment or not. While the cell survival rate of the green zoospores preserved at room temperature is equivalent to that of example 1, the cell survival rate of the chlamydospores is significantly reduced; most importantly, cells are stored at room temperature, cell agglomeration is not easy to disperse, the cells have peculiar smell, a large amount of bacteria can be observed through microscopic examination, and the growth rate is also obviously reduced after recovery.
Example 2
Haematococcus pluvialis SCCAP K-0084 strain (commercially available) in nitrogen-free BBM medium at 300. mu.E/m2Culturing algae strain under illumination for 10 days to obtain chlamydospore, standing and settling to obtain concentrated algae with cell density of 60g/LPutting the concentrated algae solution into a fresh-keeping chamber of a refrigerator for cold acclimation at 5 ℃ for 24 hours; then subpackaging in a plurality of sample bags, freezing into ice in a refrigerator at-5, -20, -80 ℃ respectively after subpackaging, and continuously freezing for 60 days. Then taking out and melting at room temperature, diluting with BBM culture medium containing 4mmol/L sodium nitrate after melting, concentrating algae solution until cell density is 0.5g/L, and placing at 200 μ E/m2Illumination is carried out per second and 1% CO is introduced2Air culture of (2). After 48 hours of illumination culture, observing and counting the proportion of the cells capable of exciting the red chlorophyll fluorescence by using a fluorescence microscope, and counting the survival rates of 98 percent, 96 percent and 16 percent at the temperatures of-5, -20 and-80 ℃.
Example 3
300 μ E/m in nitrogen-free BBM medium2Culturing the algae strain under illumination for 10 days to obtain chlamydospore algae liquid, standing and settling to obtain concentrated algae liquid with the cell density of 40g/L, and cold acclimating in a refrigerator fresh-keeping chamber at 5 ℃ for 24 hours; then the mixture is subpackaged in a plurality of sample bags and transferred to a refrigerating chamber to be frozen into ice at-5 ℃. Taking out a bag of the cryopreserved algae after the cryopreservation for 3 days, 38 days and 75 days respectively, and placing the bag at room temperature for thawing; then diluting the above concentrated solution with BBM medium containing 4mmol/L sodium nitrate until cell density is 0.5g/L, placing under 200 μ E/m2/s illumination, and introducing 1% CO2Air culture of (2). And (5) sampling at regular time and monitoring the growth condition of the cells. The experiment in which only the cells subjected to cold acclimation treatment without freezing were used as the inoculation material was used as a control. The results show that the cell survival rates of the cells recovered after 3, 38 and 75 days of cryopreservation are 100%, 98% and 96% respectively; the cell densities of the cells cultured under the above culture conditions for 8 days were 3.61, 2.86 and 2.64g/L, respectively.
Example 4
Culturing Haematococcus pluvialis in an outdoor 1000L column photoreactor for 10 days under nitrogen-free condition to obtain chlamydospore algae solution, performing gravity settling to obtain concentrated algae solution with cell density of 40g/L, and cold acclimating in a refrigerator fresh-keeping chamber at 5 deg.C for 24 hr; then frozen to ice at-5 ℃ and kept for 75 days. Then placing the mixture at room temperature for thawing, diluting the thawed concentrated algae solution by using BBM culture medium containing 4mmol/L sodium nitrate until the cell density is 0.3g/L,inoculating into 1000L column type photoreactor, culturing under outdoor natural light irradiation with 1% CO2Air culture of (2). Every two days, samples were taken and cell growth was monitored. The cell density after 12 days of culture was 2.04 g/L.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention, the parts not specifically described or shown being exaggerated for clarity of presentation and for clarity of illustration in the prior art and not in any greater detail herein. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and features disclosed herein.
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